JPH07267867A - Treating agent having antiendotoxin activity - Google Patents

Abstract

PURPOSE:To obtain the subject agent capable of efficiently neutralizing an endotoxin released by a gram-negative bacterium and useful as a treating agent for endotoxin shock, a pretreating agent for e.g. a regeneration treatment of periodontal tissue, etc. CONSTITUTION:This is a toxin inhibitor against an endotoxin of a gram-negative bacterium containing a macrolide antibiotic having a 14-membered ring, preferably at least one kind selected from an erythromycin, a clarithromycin, a triacetyloleandomycin and a roxithromycin. In the case of medical use, this inhibitor is preferably used as an oral preparation or an injection which is a directly administrable preparation, i.e., an intravenous injection and in the case of dental use, as an ointment or a film which has sustained releasability and is directly administrable into a periodontal pocket. In the case of oral preparation which is systemically administered, a preferable dose is 300-1500mg/ day and in the case of an external preparation, etc., which is topically administered, 15mg/day.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to inhibition of toxicity of endotoxin released from Gram-negative bacteria, and specifically, it is applied to periodontal disease and diseases caused by systemic endotoxin.

[0002]

BACKGROUND OF THE INVENTION Endotoxin is known as one of the components constituting the extracellular membrane of Gram-negative bacteria. Endotoxin is a kind of lipopolysaccharide (LPS), and its chemical structure is composed of a part composed of a polysaccharide and a lipid part (usually called lipid A). When the endotoxin is present in the extracellular membrane of the bacterial cell, the lipid A portion, which is the hydrophobic portion of the endotoxin, is present in the form of being embedded in the lipid bilayer membrane of the extracellular membrane, and the hydrophilic portion of the endotoxin is present. The polysaccharide moiety is present outside the cell in a form that extends. The lipid A part is responsible for the biological activity of endotoxin, and there are various types of its activity. On the other hand, it has an immunostimulatory effect and has a favorable effect on the living body.
It also has adverse effects such as causing shock symptoms.

Normally, endotoxin does not exhibit its activity when it is present in the extracellular membrane of cells, but it is released to the outside of cells when the bacteria die or, recently, when they divide. I know. In the field of medical science, a large amount of endotoxin is released into the blood in patients with serious infectious diseases, which causes shock symptoms and sometimes death, which is a serious problem. As a treatment method for such endotoxin shock, for example, as shown in Japanese Patent Application Laid-Open No. 4-213393,
Method using a monoclonal antibody and JP-A-4-5065
As shown in No. 10, many methods using a bactericidal / permeability-enhancing protein and methods using an anti-inflammatory agent have been reported.

Also in the field of dentistry, the action of endotoxin has been taken up as a problem, particularly in the treatment of periodontal disease. Periodontal disease is regarded as an infectious disease caused by bacteria (especially Gram-negative anaerobic bacteria) and is considered to be a major cause of tooth loss. In periodontal disease, endotoxin is released from the extracellular membrane of periodontopathic bacteria and acts on living cells such as gingival fibroblasts, which causes various cytokines to be expressed and also contributes to gingival inflammation and alveolar bone absorption. It is believed to have an impact. Furthermore, the problem of endotoxin in periodontal disease is its influence on the process of periodontal tissue regeneration. Normally, endotoxin is easily adsorbed on the tooth surface, and its removal can only be dealt with by physical treatment such as scaling and curtage. However, it is considered that it is difficult to completely remove it by such physical treatment alone, and its residual is well-discussed about the periodontal tissue regeneration process. Regarding the chemical treatment, a system using tauroline (Japanese Patent Application Laid-Open No. Hei 5 (1999) -58242)
-2018878) and the like, and its toxicity neutralizing action is explained. Thus, endotoxins released by Gram-negative bacteria exhibit their toxicity in both medical and dental fields, causing therapeutic problems. Therefore, it is desired to develop a drug that can efficiently neutralize the toxicity of endotoxin.

[0005]

[Means for Solving the Problems] As a result of intensive studies from the above viewpoints, the present inventors have found the effect of neutralizing the toxicity of endotoxin in macrolide antibiotics and completed the present invention. It is a thing. Macrolide antibiotics used in this method include acetylspiramycins, erythromycins, triacetyloleandomycins, acetylquitasamycins, clarithromycins, midecamycin acetates, midecamycins, josamycins, Examples include spiramycins, roxithromycins, and rokitamycins. Of the macrolide antibiotics, macrolide antibiotics having a 14-membered ring are preferable, and erythromycins, clarithromycins, triacetyloleandomycins, and roxithromycins are particularly preferable. The most preferred macrolide antibiotics are erythromycin, triacetyloleandomycin, clarithromycin, roxithromycin. These antibiotics may be used alone or in combination of two or more. The dosage form and administration method of these macrolide antibiotics are preferably the oral administration method or the direct administration dosage form such as intravenous administration, intramuscular administration and subcutaneous administration when administered for medical use. On the other hand, when it is used for dentistry, an oral administration method or a parenteral administration method may be used, but it is preferable to use an ointment or a film agent which can be directly administered into the periodontal pocket and has a sustained release property.

The dose of the macrolide antibiotic in the present invention varies depending on its administration form and the degree of disease, but it is 200 to 2000 mg / day orally by systemic administration.
The dose is preferably 300 to 1500 mg / day, and 0.5 to 10 mg / day, preferably 1 to 5 mg / day in the case of a topical drug for topical administration. For topical administration,
It is possible to further reduce the daily dose by combining it with a base having sustained release properties. In particular, when a local toxicity alleviation effect is expected, such as in periodontal disease, a smaller dose than a macrolide antibiotic is usually administered for sterilization, and it is effective in terms of safety such as side effects. In terms of aspects, it is highly useful.

[0007]

EXAMPLES The present invention will be described in detail below with reference to examples. However, the present application is not limited to these examples. Test Example 1: LPs of periodontopathic bacteria and Escherichia coli shown below
Prepare S and antibiotics in a weight ratio of 1: 2. In short, L
A suspension of PS in distilled water (10 μg / ml) was mixed with 1 ml of an antibiotic solution (20 μg / ml), and the mixture was incubated at 37 ° for 2 hours. This solution was appropriately diluted, and the LPS activity was measured by the Limulus test. The judgment criteria in the table are that the suppression of the activity value is markedly suppressed (50% or more) ++,
Suppression (20-50%) is indicated by + and no effect is indicated by-.

[0008]

[Table 1]

Test Example 2: The effect was confirmed by the survival rate after administration of endotoxin in ICR male mice. E. coli LPS (manufactured by Difco) as endotoxin and independently prepared Porphyromonas gingivalis (P.
g) and L of Prevotella Intermedia (P.i)
PS was used. The doses of E. coli (500 μg) and others (1500 μg) were suspended in physiological saline and administered via the tail vein of mice. Each drug was intraperitoneally administered at 50 mg / kg. Hydrocortisone (100 mg / k as a control drug)
g) was used. The survival rate on the 5th day is shown.

[0010]

[Table 2]

Test Example 3: It was confirmed whether the effect of LPS on the proliferation of human periodontal ligament (PDL) fibroblasts and its toxic effect could be inhibited by macrolide antibiotics. PDL fibroblasts were isolated from the periodontal ligament attached to the tooth bud, which was expediently removed from a patient with a healthy periodontal tissue, and primary cultured, and Dulbecco's modified Eagle MEM medium containing 10% fetal bovine serum (FBS) ( Cells (passage number 6 to 10) subcultured and cultured in DMEM were used. P that reached logarithmic growth phase
3 × 10 4 DL cells were planted in a Petri dish having a diameter of 35 mm and cultured in DMEM / 10% FBS medium. After 24 hours, the cells were washed with PBS (-), replaced with DMEM / 1% FBS medium containing LPS and 20 µg / ml of antibiotics so that the final concentration was 10 µg / ml, and the culture was further continued. Erythromycin, clarithromycin, polymyxin B, and streptomycin were used as antibiotics.
As a control, culture was performed in a medium containing no antibiotic. 48 hours after the medium exchange, the cells were detached with 0.15% trypsin / 0.1% EDTA and collected, and the cells were counted with a hemocytometer. In addition, LPS is P. g, P. i was used. The results are shown in Table 3. Judgment is ++ when the growth suppression is 20% or less, and +5 when the growth suppression is 20 to 50%.
Those exceeding 0% are indicated by-.

[0012]

[Table 3]

Example 1 A stock gel is prepared by swelling carboxyvinyl polymer, hydroxypropylmethyl cellulose and polyvinyl acetic acid as a drug layer in water. Take an appropriate amount of these, add erythromycin dissolved in ethanol, and mix well. The obtained gel is spread, dried (75 ° C., 10 min), and then cut. Separately, a methanol solution of polyvinyl acetic acid was spread on a release paper as a support layer, dried (75 ° C., 8 min), and then cut. The drug layer and the support layer were thermocompression bonded with an iron (110 ° C,
30 sec) It was cut into a size of 1 × 2 cm to obtain a desired film-form pharmaceutical preparation.

Ingredients Mixing ratio (weight) (Drug layer) Erythromycin 2 parts Carboxyvinyl polymer 20 parts Polyvinyl acetate 50 parts Hydroxypropylmethyl cellulose 30 parts Sodium citrate 1.5 parts (Support layer) Polyvinyl acetic acid 100 parts It is used in the field of dentistry and attached to the gingiva. It can also be used by inserting it into the periodontal pocket.

Example 2 Glycerin and hydroxyethyl cellulose are kneaded to prepare a non-aqueous gel. Clarithromycin, Eudragit RS, and triacetin were added thereto to obtain a pharmaceutical preparation having a desired sustained release property. Ingredients Mixing ratio (weight) Clarithromycin 2 parts Hydroxyethylcellulose 4 parts Eudragit RS 2 parts Triacetin 10 parts Concentrated glycerin 82 parts In this example, the gingiva was directly applied or injected into a periodontal pocket using a dental syringe. use.

[0016]

INDUSTRIAL APPLICABILITY According to the method of the present invention, endotoxin can be efficiently neutralized, and it can be used as a pretreatment agent such as a therapeutic agent for endotoxin shock in the medical field, a periodontal tissue regeneration procedure in dentistry and the like. it can.

Claims (6)

[Claims] 1. An endotoxin toxicity inhibitor of Gram-negative bacteria, which comprises a macrolide antibiotic. 2. The inhibitor according to claim 1, wherein the macrolide antibiotic is a macrolide antibiotic having a 14-membered ring. 3. The inhibitor according to claim 1, wherein the macrolide antibiotic is one or more selected from erythromycins, clarithromycins, triacetyloleandomycins and roxithromycins. . 4. The inhibitor according to claim 1, which is an oral agent or an injectable agent. 5. The inhibitor according to claim 1, which is a therapeutic agent for periodontal disease. 6. The inhibitor according to claim 5, wherein the therapeutic agent is a preparation which is directly administered into the periodontal pocket and penetrates or stays inside the pocket.